Gene expression is important for understanding a wide range of biological phenomena, including development, differentiation, senescence, oncogenesis, and many other medically important processes. Recently, changes in gene expression have also been used to assess the activity of new drug candidates and to identify new targets for drug development. The latter objective is accomplished by correlating the expression of a gene or genes known to be affected by a particular drug with the expression profile of other genes of unknown function when exposed to that same drug; genes of unknown function that exhibit the same pattern of regulation, or signature, in response to the drug are likely to represent novel targets for pharmaceutical development.
Generally, the level of expression of the protein product of a gene and its messenger RNA (mRNA) transcript are correlated, so that measuring one provides you with reliable information about the other. Since in most instances it is technically easier to measure RNA than to measure protein, variations in mRNA levels are commonly employed to assess gene expression in different cells and tissues or in the same cells and tissues at different stages of disease or development or exposed to different stimuli. One particularly useful method of assaying gene expression at the level of transcription employs DNA microarrays (Ramsay et al., Nature Biotechnol. (1998) 16:40-44; Marshall and Hodgson, Nature Biotechnol. (1998) 16:27-31; Lashkari et al., P.N.A.S. USA (1997) 94:130-157; and DeRisi et al., Science (1997) 278:680-686).
Mammalian cells contain as many as 1×105 to 3×105 different mRNA molecules, each of which varies in abundance (or frequency) within a given cell. The most abundant mRNAs are typically present at many thousands of copies per cell, while others may be present in as few as one copy or less per cell. Techniques for analyzing gene expression at the level of transcription typically require tens to hundreds of micrograms of mRNA, or as much as might be found in 107-109 mammalian cells. Oftentimes, it is impractical to obtain this number of cells from a tissue of interest. For example, a blood sample typically contains 106 nucleated cells/ml; hence, to obtain 109 cells for analysis would necessitate taking a 1000 ml blood sample, which is clearly impractical in most instances.
Various methods have been described in the literature for amplifying the amount of a nucleic acid, such as deoxyribonucleic acid (DNA) and RNA, present in a sample. Among these, the most widely practiced is the polymerase chain reaction (PCR), described in U.S. Pat. No. 4,683,195 by Mullis et al., entitled “Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences,” issued 28 Jul. 1987 and U.S. Pat. No. 4,683,202 by Mullis, entitled “Process for Amplifying Nucleic Acid Sequences,” issued 28 Jul. 1987, and herein incorporated by reference. Briefly, PCR consists of amplifying denatured, complementary strands of target nucleic acid by annealing each strand to a short oligonucleotide primer, wherein the primers are chosen so as to flank the sequence of interest. The primers are then extended by a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence. Each successive cycle of denaturation, primer annealing, and primer extension essentially doubles the amount of target synthesized in the previous cycle, resulting in exponential accumulation of the target.
PCR methodologies in general suffer from several limitations that are well known in the art; see U.S. Pat. No. 5,716,785 by Van Gelder et al., entitled “Processes for Genetic Manipulations Using Promoters,” issued 10 Feb. 1998) and PCR Protocols: A Guide to Methods and Applications, (Innis et al. (Ed.) Academic Press Inc., San Diego, Calif., 1990) for review and discussion of limitations. One such limitation is that different cDNAs may be amplified with different efficiencies, resulting in under-representation of some cDNA sequences and overrepresentation of others in the amplified product. Even a small difference in efficiency may result in a several-thousand fold differential in the representation of these cDNAs in the product after only thirty cycles of amplification. An attempt at using PCR to globally amplify all cDNAs in a sample for gene expression profiling reported an unacceptable degree of variability.
An alternative method of mRNA amplification is known as in vitro transcription (IVT) and is described in U.S. Pat. No. 5,716,785 by Van Gelder et al., entitled “Processes for Genetic Manipulations Using Promoters,” issued 10 Feb. 1998, which is herein incorporated by reference. Unlike PCR, IVT does not result in geometric amplification, but rather in linear amplification. In IVT, an oligo(dT) primer that is extended at the 5′-end with a bacteriophage T7 RNA polymerase promoter (RNAP) is used to prime the poly-A+ mRNA population for cDNA synthesis. After synthesis of the first-strand cDNA, the second-strand cDNA is made using the method of Gubler and Hoffman (Gene (1983) 25:263-269). Addition of RNA polymerase results in in vitro transcription and linear amplification of mRNA that is anti-sense to the poly-A+ RNA.
Two other types of nucleotide amplification are described in Stoflet et al., Science (1988) 239:491-494) and in Sarkar and Sommer, Science (1989) 244:331-334). Stoflet et al. describes a variation of PCR known as genomic amplification with transcript sequencing (GAWTS). In GAWTS, a phage promoter is attached to at least one of the two PCR primers. The DNA segments amplified by PCR are transcribed to further increase signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. Sarkar and Sommer (1989) supra, describes a modification of the GAWTS method, known as RNA amplification with transcript sequencing (RAWTS). RAWTS consists of four steps:                (i) cDNA synthesis with oligo(dT) or an mRNA-specific oligonucleotide primer,        (ii) PCR where one or both oligonucleotides contains a phage promoter attached to a sequence complementary to the region to be amplified,        (iii) transcription with a phage promoter, and        (iv) reverse transcriptase-mediated dideoxy sequencing of the transcript, which is primed with a nested (internal) oligonucleotide.        
Both the GAWTS and RAWTS methods, however, involve relatively large numbers of PCR cycles, e.g. twenty-seven rounds (Stoflet et al. (1988) supra) or forty rounds (Sarkar and Sommer (1989) supra). As described above, large numbers of PCR cycles suffer from different efficiencies of amplification, and ensuing variability
There exists a need in the art for improved methods of amplifying nucleic acids, especially mRNA, which methods can achieve a high degree of amplification from a limited amount of mRNA and which are reproducible. New improved methods for amplifying nucleic acids are also needed in the art that reduce the time required to perform the amplification process and greatly improve the efficiency of amplification. Such new improved methods would greatly facilitate the rapid processing of clinical samples, and would also be amenable to automation, which would provide substantial commercial advantages for medical and diagnostic applications. The present invention is believed to satisfy these needs and to provide other related advantages.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.